The effect of the traditional medicine phela on p-glycoprotein and multidrug resistance-associated protein 2 drug transporters in the gastrointestinal tract of a rat model

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THE EFFECT OF THE TRADITIONAL MEDICINE PHELA

ON P-GLYCOPROTEIN AND MULTIDRUG

RESISTANCE-ASSOCIATED PROTEIN 2 DRUG

TRANSPORTERS IN THE GASTROINTESTINAL TRACT

OF A RAT MODEL

BY

MOLEBOHENG EMILY BINYANE

(B.Sc. Microbiology, B-Tech Project Management, B.Med.Sc. Hons

Pharmacology)

A dissertation submitted in fulfilment of the requirements in respect of the

Master of Medical Science (M.Med.Sc) degree in Pharmacology

in the Faculty of Health Sciences

at the University of the Free State

Supervisor: Prof. A. Walubo

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ABSTRACT

Phela is the herbal preparation of four African traditional medicinal plants, and is under the development by the Medical Research Council (MRC) as an immune stimulant for immune compromised individuals. Patients might use Phela with other medicines; therefore, the herb-drug interactions profiling of Phela is important. Membrane drug-transporters such as P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) are considered important factors in determining the pharmacokinetic parameters of drugs such as paclitaxel (PTX) and methotrexate (MTX), respectively. Inhibition or induction of transport might result in drug interactions with other drugs transported by these respective transporters. Moreover, significant herb-drug interactions involving P-gp and MRP2 have been described. Therefore, the effect of Phela on P-gp and MRP2 in the gastrointestinal tract of a rat model was investigated here.

First, a high performance liquid chromatography (HPLC) method for determination of PTX in plasma was developed. It involved liquid-liquid extraction of 100 µl plasma, spiked with PTX, extracted with diethyl ether: dichloromethane (2:1), followed by centrifugation. The supernatant was evaporated to dryness under a stream of nitrogen, reconstituted, and 100 µl was injected into the HPLC. The sample was eluted with a mobile phase of sodium phosphate buffer (pH 2): acetonitrile (60:40, v/v) over a C8 (1)

(4.6 X 250 mm) 5 µ analytic column at 1 ml/min. PTX was detected by UV at 230 nm. Docetaxel (DTX) was used as the internal standard. Under these conditions, DTX and PTX eluted at retention times of 6.595 and 6.038 minutes, respectively. The average calibration curve (0-15 µg/ml) was linear with a regression equation of y = 0.1931x + 0.0705, and correlation coefficient (r) of 0.9973. The method was used successfully in animal experiments to measure PTX in the plasma of treated rats.

Thereafter, a preliminary experiment was conducted in vitro to establish whether Phela has a direct/ physical effect on PTX, using a direct drug interaction testing experiment in buffer, as well as Slide-A-Lyzer®_{dialysis. During the direct drug interaction experiment,}

buffer was spiked with 10 µg/ml of PTX with or without 3.85 mg/ml Phela, and PTX concentrations were determined by HPLC. Then, using a Slide-A-Lyzer®_dialysis

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cassette, the time of equilibrium of PTX was determined by monitoring the changes in PTX concentrations over 12 hours, in plasma containing 230 µg/ml PTX and buffer. Thereafter, the potential of an interaction was tested by adding 88.55 mg/ml Phela to the same experiment after 8 hours of incubation, and monitoring PTX concentrations after 10 and 12 hours by HPLC. In the first experiment, Phela had no direct effect on PTX concentrations, while in the second experiment the time of equilibrium of PTX was estimated at 8 hours. After Phela was added, PTX concentrations and its free fraction (fu) remained unchanged. Therefore, it was concluded that there is no interaction between Phela and PTX in vitro.

This final part of the study was undertaken to investigate the effect of Phela on P-gp and MRP2 transporters. PTX and cyclosporin A (CyA) were used as the respective substrate and inhibitor of P-gp, while MTX and probenecid (PRO) were those of MRP2. Ethical approval was obtained and male Sprague-Dawley (SD) rats (200-250 g) were used. The animal experiment was divided into two parts. In Part I, three groups of 40 rats each received a one-off oral dose of PTX-only (10 mg/kg); PTX & CyA (10 mg/kg); or PTX & Phela (15.4 mg/kg), while in Part II, three groups of 40 rats each received a one-off oral dose of MTX-only (10 mg/kg); MTX & PRO (20 mg/kg); or MTX & Phela (15.4 mg/kg). For each group, 5 rats were sacrificed after 0.5, 1, 2, 4, 6, 8, 10, and 12 hours. Blood was analysed for full blood count, liver function, and PTX and MTX concentrations. CyA and PRO increased the area under the plasma concentration-time curve (AUC) of PTX and MTX, respectively, whereas Phela had no effect on the AUC of PTX or MTX.

Overall, no direct interaction between PTX and Phela was observed both in vitro and in vivo, and there were also no interactions between MTX and Phela in vivo. Phela did not inhibit P-gp or MRP2. This implies that Phela will most probably not be involved in herb- drug interactions of membrane transporter origin. Therefore, the doses of drugs that are transported by P-gp and MRP2 need not be adjusted when co-administered with Phela.

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DECLARATION OF INDEPENDENT WORK

I, Moleboheng Emily Binyane, hereby declare that the master’s research dissertation that I herewith submit at the University of the Free State, is my own independent work and that I have not previously submitted it for a qualification at another institution of higher education. I am aware that the copyright is vested in the University of the Free State.

10 November 2015

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SUPERVISOR’S DECLARATION

I, Professor A. Walubo, the supervisor of this dissertation entitled: The effect of the traditional medicine Phela on P-glycoprotein and multidrug resistance-associated protein 2 drug-transporters in the gastrointestinal tract of a rat model, hereby certify that the work in this project was done by Moleboheng Emily Binyane at the department of Pharmacology, University of the Free State.

I hereby approve submission of this dissertation and also affirm that it has not been submitted previously to this or any other institution for admission to a degree or any other qualification.

10 November 2015

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ACKNOWLEDGEMENTS

My research journey has been the most challenging academic endeavour, and without the support of the following people, this study would not have been completed. It is to all of them that I owe my deepest gratitude.

 My supervisor, Professor Andrew Walubo, who constantly advised, guided and encouraged me throughout the period of the study. His wisdom and knowledge has inspired me to work harder.

 Ms Makhotso Lekhooa, for her undying support and patience. She guided and devotedly corrected me throughout the entire study. Her presence made my journey easy.

 Mrs Zanelle Bekker, for her support and timely ordering of drugs and chemicals. She has been consistent and competent in her work and has motivated me to be the best I can.

 Dr Jan du Plessis, for the technical expertise that he offered during the method development stage of the study.

 Mr. Seb Lambrecht and staff of the animal house of the University of Free State, for their timely assistance with animals throughout the animal experiments.

 Dr. Rolien Smith, Dr. Paulina van Zyl, Mrs Refuoe Baleni and all staff members of the Department of Pharmacology, for their support during the study period.

 Faculty of Health Sciences, for the bursary which was awarded to me and the Department of Pharmacology, for fully financing the study.

 A very special thank you to my sisters; Mampho and Ntsoaki, family and friends, for their ongoing support, encouragement and understanding. Their love has kept me going.

I dedicate this work to my late father and mother, Pule and Nkutloang Binyane. They worked hard to see me succeed. Even though they are no more, their love and teachings have kept me firm.

Lastly and foremost, I thank Almighty God, Jehovah Jireh, for making this journey possible and successful!

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ABBREVIATIONS

ABC ATP-binding cassette

ALP alkaline phosphatase

ALT alanine transaminase

AST aspartate aminotransferase

AUC area under the curve

BCRP breast cancer resistance protein

Cal calibration

CLp clearance

Cmax maximum concentration

CV coefficient of variation

CyA cyclosporin A

CYP2C8 cytochrome P450 enzyme 2C8 CYP2C19 cytochrome P450 enzyme 2C19 CYP3A4 cytochrome P450 enzyme 3A4

CYP450 cytochrome P450

EMI enzyme multiplied immunoassay

FPIA fluorescence polarization immunoassay

GIT gastrointestinal tract

HPLC high performance liquid chromatography

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Ke elimination rate constant

LC-MS-MS liquid chromatography tandem mass spectrometry MCH mean corpuscular hemoglobin

MCHC mean corpuscular haemoglobin concentration

MCV mean corpuscular volume

MDR multidrug resistant

MRC Medical Research Council

MRP2 multidrug resistance-associated protein 2

MRT mean residence time

MTX methotrexatre

OATP1B1 organic anion-transporting polypeptide 1B1 P-gp P-glycoprotein

PRO probenecid

PTX paclitaxel

RIA radioimmunoassay

T½ half-life

Tmax time to reach maximum concentration

UV ultraviolet

Vd volume of distribution

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LIST OF FIGURES

Figure 2.1 Cross section and structure of the small intestine ... 4

Figure 2.2 Illustration of solute carriers (SLC), ATP-dependent carriers (ABC carriers), and channels in the plasma membrane ... 5

Figure 2.3 Illustration of organ distribution of transport proteins ... 6

Figure 2.4 The chemical structure of paclitaxel ... 9

Figure 2.5 The chemical structure of cyclosporin A ... 11

Figure 2.6 The chemical structure of methotrexate ... 13

Figure 2.7 The chemical structure of probenecid ... 14

Figure 2.8 Phela extract powder ... 18

Figure 5.1a UV-spectrum of mobile phase ... 29

Figure 5.1b UV-spectrum of paclitaxel ... 30

Figure 5.1c UV-spectrum of docetaxel ... 30

Figure 5.2a Chromatogram of mobile phase ... 31

Figure 5.2b Chromatogram of mobile phase spiked with paclitaxel ... 31

Figure 5.2c Chromatogram of mobile phase spiked with paclitaxel and docetaxel ... 31

Figure 5.2d Chromatogram of a blank plasma sample ... 32

Figure 5.2e Chromatogram of a plasma sample spiked with 10 µg/ml paclitaxel and 10 µg/ml docetaxel ... 32

Figure 5.3 Average 5 day calibration curve of paclitaxel ... 33

Figure 5.4a Chromatogram of blank rat plasma ... 36

Figure 5.4b Chromatogram of paclitaxel (0.74 µg/ml) in rat plasma after 8 hours of 10 mg/kg one-off oral administration ... 36

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Figure 6.2 Plot of time of equilibrium of paclitaxel ... 43

Figure 6.3 Plot of paclitaxel concentrations after the addition of Phela ... 45

Figure 7.1 A schematic illustration of Part I of the animal experiment ... 50

Figure 7.2 A schematic illustration of the preliminary experiment of Part II of the animal experiment ... 53

Figure 7.3 A schematic illustration of Part II of the animal experiment ... 55

Figure 7.4 A photograph of blood collection via cardiac puncture ... 57

Figure 7.5 Paclitaxel concentrations after treatment with Phela over 12 hours ... 61

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LIST OF TABLES

Table 2.1 Herb drug interactions mediated by drug metabolising enzymes or

transporters ... 17

Table 5.1 HPLC calibrations for paclitaxel using ratio paclitaxel/ ratio docetal ... 33 Table 5.2 Summary of accuracy data of paclitaxel in plasma at 1.25, 7.50 and 15.00

µg/ml paclitaxel ... 34

Table 5.3 Summary of short and long term stability data of 1.25, 7.50 and 15.00

µg/ml paclitaxel in plasma at ambient temperature, 4 °C and -20 °C measured after 24 and 48 hours and 1, 2, and 4 weeks ... 35

Table 6.1 Average (mean±SD) paclitaxel concentrations after the direct buffer

experiment ... 42

Table 6.2 Average (mean±SD) paclitaxel concentrations and its free fraction

percentage ... 43

Table 6.3 Average (mean±SD) paclitaxel concentrations with Phela, and its free

fraction percentage... 44

Table 7.1 Average (mean±SD) methotrexate concentrations after 0.5 and 4 hours of

co-treatment with probenecid ... 52

Table 7.2 Average (mean±SD) full blood count and platelets results of the PTX-only,

PTX & CyA, and PTX & Phela groups over 12 hours ... 59

Table 7.3 Average (mean±SD) liver function tests results of PTX-only, PTX &

CyA, and PTX & Phela groups over 12 hours ... 60

Table 7.4 Average (mean±SD) paclitaxel concentrations after 12 hours of

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Table 7.5 Paclitaxel pharmacokinetic parameters in rats after 12 hours of

treatment with Phela... 62

Table 7.6 Average (mean±SD) full blood count and platelets results of the MTX-only,

MTX & PRO, and MTX & Phela groups over 12 hours ... 63

Table 7.7 Average (mean±SD) liver function tests results of MTX-only, MTX & PRO,

and MTX & Phela groups over 12 hours ... 64

Table 7.8 Average (mean±SD) methotrexate concentrations after 12 hours of

co-treatment with Phela ... 65

Table 7.9 Methotrexate pharmacokinetic parameters in rats after 12 hours of

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CHAPTER 1 GENERAL INTRODUCTION

Research has taken interest in membrane transporters, particularly because of their role in determining pharmacokinetic, safety and efficacy profiles of drugs (Giacomini and Sugiyama, 2006). Membrane transporters are membrane-associated proteins that govern the transport of influx and efflux ions, nutrients, and drugs (Huang et al., 2004). In particular, more than 400 membrane transporters in two major super families, i.e., ATP-binding cassette (ABC) and solute carrier (SLC), have been described in the human genome. Many different drug-transporters are expressed in various tissues, such as the epithelial cells of the intestine and kidney, hepatocytes, and brain capillary endothelial cells (Takano et al., 2006).Transporters can play a vital role in determining drug concentrations in the systemic circulation, as well as in cells.

It is becoming increasingly evident that, among other transporters, the intestinal transporters play an important role in the oral absorption of compounds, with both influx and efflux transporters influencing drug absorption processes (Pang, 2003).Oral absorption of compounds can be limited by efflux transporters located in the intestine, such as P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (MRP2) (Chan et al., 2004), while influx transporters such as the organic anion-transporting polypeptide (OATP) and peptide transporters (e.g., PEPT1), can aid intestinal drug absorption (Kim, 2003). Moreover, there appears to be an overlap in the substrate specificity between the efflux transporter P-gp and the influx transporter OATP, which

could lead to opposing influences on the net absorption of a shared substrate (Kim, 2003).

Most known interactions between herbal extracts and drugs involve the inhibition of drug-metabolising enzymes, but little is yet known about the possible role of transporters in these interactions (Fuchikami et al., 2006). Citrus juices, including grapefruit juice, have been reported to reduce the bioavailability of orally administered fexofenadine. This interaction is considered to be caused by the inhibition of intestinal OATPs (Banfield et al., 2002). This decrease in exposure could result in reduced efficacy of

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fexofenadine in patients (Kamath et al., 2005). Therefore, it is necessary to screen traditional medicines for their effects on drug-transporters involved in drug absorption. In this study, more interest was placed particularly on Phela, a traditional medicine. Phela is the herbal mixture of four African traditional medicinal plants that has been used for decades in wasting conditions and, after successful observation studies in humans, is now being developed by the Medical Research Council (MRC) as an immune booster for patients with a compromised immune system (Lekhooa et al., 2012). Due to the fact that Phela may be used by HIV patients who may be using other medications as well, it was therefore important to create a model to provide a method of predicting possible drug interactions of Phela. Here, Phela was screened for potential interaction with two efflux drug-transporters, P-gp and MRP2, involved in drug absorption, with a hope that this developed technique can also be applied for screening other products in development.

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CHAPTER 2 LITERATURE REVIEW: PART I

AN OVERVIEW OF MEMBRANE TRANSPORTERS

2.1 Drug absorption

Absorption is the movement of a drug from the site of administration to the circulation, where the route of administration is the determinant of the rate and efficiency of absorption. When a drug is administered intravenously, the total dose is able to reach the bloodstream (i.e., absorption is complete), whereas an orally administered drug may undergo partial absorption which results in low bioavailability (Richard et al., 2009, p.7-8).

Although oral administration of drugs is more convenient and acceptable to patients, good oral bioavailability is important because it means the total drug can reach the circulation via the gastrointestinal tract. Therefore oral drug absorption is continuously being researched in order to improve bioavailability, taking into consideration both the kinetics and dynamics of the orally administered drug (Pang, 2003).

Due to their major role in first-pass metabolism, the liver and small intestine are important in the absorption of orally administered drugs. The small intestine has a large surface area and facilitates the majority of drug absorption due to the presence of villi and microvilli (Figure 2.1, letters B and C), which increases the absorptive area. The highest concentration of villi and microvilli is located in the duodenum and jejunum, while it is least in the ileum. Furthermore, the circulation of the intestine is unique, in that it is the vesicular portal system with which drugs are delivered to the liver.

Drug delivery rate and the extent of saturability of intestinal enzymes are affected by the amount of drug entering the intestine, as well as by the rate of blood flow. This eventually affects the rate of intestinal and hepatic first-pass metabolism. Moreover, absorption is also influenced by other variables such as drugs or food (Pang, 2003).

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Figure 2.1: Cross section and structure of the small intestine (From:

http://wwww.daviddarlinginfo/encyclopedia/copyright.htmlcited26/07/11)

Though it is believed that most drugs are absorbed by a simple diffusion mechanism via the gastrointestinal epithelium, direct and indirect evidence points to the participation of transporters in mediating absorption (Tsuji, 2006). Here, it was found that membrane- bound drug-transporters have absorbed or secreted several drugs that had poor bioavailability. Subsequently, this shifted the focus to the involvement of membrane transporters in the mechanism of drug absorption.

Transporters that are expressed in the intestines of humans are also found in rats (Mizuno et al., 2003), meaning that transporters involved in drug absorption are found in the intestines of both humans and rats, therefore a rat model was appropriate for the purpose of this study.

2.2 Membrane transporters

Membrane transporters are defined as membrane-associated proteins responsible for the transport of solutes, including drugs and other xenobiotics, into and out of cells (Giacomini et al., 2010). Transporters are key determinants of drug concentrations in the bloodstream and in cells. There are two major super-families of membrane transporters,

B

C

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namely: the ATP-binding cassette (ABC) and solute carrier (SLC) transporters (Figure 2.2).In order to understand the molecular characteristics of individual transporters belonging to these families, many transporters have been cloned and studied, thus extensive progress has been made. It is now known that some of these transporters govern drug transport in different tissues, and they may become main determinants of the pharmacokinetic characteristics of a drug as far as its intestinal absorption, tissue distribution, and elimination are concerned (Giacomini et al., 2010).

Figure 2.2: Illustration of solute carriers (SLC), ATP-dependent carriers

(ABC-carriers), and channels in the plasma membrane (From: Mizuno et al., 2003).

Different drug-transporters are located in various tissues (Figure 2.3), such as the epithelial cells of the intestine and kidney, hepatocytes, and brain capillary endothelial cells (Mizuno et al., 2003). Transporters have been grouped as primary, secondary, or tertiary active transporters. Primary active transporters include: ATP-binding cassette

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transporters, such as multidrug resistant (MDR), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP), which are driven by the force of ATP hydrolysis. They are responsible for the efflux of potentially toxic endogenous and exogenous compounds from cells (Vlaming et al., 2011). Secondary active transporters include: organic anion transporter (OAT), organic anion-transporting polypeptide (OATP), sodium taurocholate co-transporting peptide (NCTP), organic cation transporter (OCT), and oligopeptide transporter (PEPT), which are driven by an exchange or co-transport of intracellular and/or extracellular ions (Mizuno et al., 2003).

Figure 2.3: Illustration of organ distribution of transport proteins (From: Tsuji, 2006)

For purposes of this study, two primary active efflux membrane drug-transporters, multidrug resistant and multidrug resistance-associated protein, involved in absorption in the gastrointestinal tract, were reviewed.

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2.2.1 P-glycoprotein

P-glycoprotein (P-gp), also known as multidrug resistant, is a type of ATPase, energy-dependent trans-membrane drug efflux pump which is one of the ABC transporters.

Furthermore, P-gp is a glycoprotein that has a molecular weight of approximately 170 kDa. It appears as a single chain with two equal homologous portions, both

containing six trans-membrane domains and two ATP-binding areas divided by an elastic linker polypeptide area between the Walker A and B motifs (Varma et al., 2003). P-gp was the first discovered transporter due to its ability to confer multidrug resistance to cancer cells (Juliano and Ling, 1976). Additionally, P-gp facilitates the ATP-dependent export of drugs from cells to the blood-stream. In the intestine, P-gp is situated in the apical membrane of mature enterocytes (ƠBrien and Cordon-Cardo, 1996), where it mediates the transport of substrates out of the cell into the intestinal lumen, thereby forming a barrier to drug absorption. The level of expression and function of P-gp can be changed by inhibition and induction of the transporter itself, which can affect the pharmacokinetics, efficacy or tissue levels of P-gp substrates (Zhou, 2008). Paclitaxel is a known substrate of P-gp (Nakajima et al., 2005), whereas cyclosporine (also known as cyclosporin A), verapamil, tamoxifen, quinidine, and phenothiazines are inhibitors of P-gp (Fisher and Sikic, 1995).

2.2.2 Multidrug resistance-associated protein 2

Multidrug resistance-associated protein 2 (MRP2) is a member of the ATP-binding cassette (ABC) family and is found mainly in the liver, kidneys and gut (Chen et al., 2002). MRP2 is expressed on the brush-border membrane of intestinal enterocytes, and excretes its substrates into the lumen, thereby limiting absorption (Taipalensuu et al., 2001). Methotrexate and irinotecan are examples of substrates of MRP2, as are the glucuronide conjugates of paracetamol (Chen et al., 2002). Probenecid is a known inhibitor of several transporters, and among those are the multidrug resistance-associated proteins (MRPs; Tunblad et al., 2003).

2.2.3 Role of efflux transporters in oral drug absorption

Oral administration is the predominant route for drug administration since it is convenient (Chan et al., 2004). However, in the intestine, drug-transporter interactions involving the efflux transporters often result in poor absorption and low oral

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bioavailability, as the drug is effluxed back into the intestinal lumen and subsequently excreted (Mitchell and Thompson, 2013).This means that absorption of orally administered compounds can be limited by efflux transporters such as P-gp and MRP2 (Kamath et al., 2005).

2.2.4 Efflux transporters inhibition associated interactions

Much attention has been paid to transporter-mediated processes, since these significantly modulate drug absorption, distribution, metabolism and excretion. Transporters are common sites for drug-drug interactions, as well as interactions of drugs with endogenous substrates, leading to drug toxicity and various adverse effects (Glavinas et al., 2004). As such, one drug which interacts with a transporter might inhibit the transport of another drug, either in a competitive or in a non-competitive manner. Should drug transport be inhibited, it usually results in increased bioavailability and decreased clearance, thereby markedly elevating the area under curve (AUC) of the affected drug (Stieger et al., 2000).For example, it was observed that orally administered verapamil increases the peak plasma level, prolongs the elimination half-life and increases the volume of distribution of orally administered doxorubicin due to inhibition of P-gp (Kerr et al., 1986), while co-administration of tenofovir and didanosine increased the AUC of didanosine by 40 to 60 % due to MRP2 inhibition (Weiss et al., 2007). Therefore, these transporters play an integral role in drug absorption, and should always be borne in mind when developing newly discovered drugs. However, there is few data on the effects of herbal medicinal compounds on these drug transporters.

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CHAPTER 2 LITERATURE REVIEW: PART II

SUBSTRATES AND INHIBITORS OF P-GLYCOPROTEIN AND

MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 2

TRANSPORTERS

2.3 Substrates and inhibitors of P-glycoprotein

2.3.1 Paclitaxel

Paclitaxel is an antineoplastic agent which belongs to a group of cytotoxic agents, the taxanes (Martin et al., 1998). Its main mechanism of action is mediated by the stabilization of cellular microtubules, and investigators have demonstrated paclitaxel activity against adult epithelial ovarian cancer, breast cancer and melanoma (Eric et al., 1995). During treatment, drug-related hypersensitivity reactions may occur (Britten et al., 2000). Nonetheless, paclitaxel has been identified as a substrate of P-glycoprotein (P-gp; Sparreboom et al., 1997).

2.3.1.1 Physical properties

Paclitaxel (Figure 2.4) is a white to off-white crystalline powder with an empirical formula of C47H51NO14 and molecular weight of 853.9 g/mol. It is slightly soluble in water, and melts

between 216-217°C (Kumar et al., 2009). Furthermore, the drug is available in an intravenous formulation, namely Taxol® (Kumar et al., 2009).

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2.3.1.2 Pharmacokinetics

Paclitaxel is administered intravenously since the oral formulation is considered problematic due to its poor absorption. Paclitaxel is widely distributed in the body and 95 % to 98 % is bound by plasma proteins, primarily albumin (Choi and Li, 2005).The metabolism of paclitaxel is catalyzed by cytochrome P450 (CYP) enzymes. Here, CYP2C8 is responsible for the formation of 6α-hydroxypaclitaxel, whereas the formation of 3’-p-hydroxypaclitaxel is catalyzed by CYP3A4 (Harris et al., 1994), and 6α3’-p-dihydroxypaclitaxel is formed after stepwise hydroxylations by CYP2C8 and CYP3A4. The primary route of elimination of paclitaxel is by hepatic metabolism and biliary excretion (Monsarrat et al., 1993).

2.3.1.3 Drug interactions

A number of clinically important drug interactions have been reported for paclitaxel. Firstly, co-administration of paclitaxel with lapatinib results in decreased clearance of lapatinib due to the inhibition of CYP3A4, CYP2C8 and P-gp. Also, cyclosporin A increases the absorption of paclitaxel by effectively blocking P-gp (Terwogt et al., 1999), while valspodar (an analogue of cyclosporin D) increases brain levels of paclitaxel by potently inhibiting P-gp as well. Furthermore, the total body clearance of paclitaxel and digoxin has been found to decrease substantially after co-treatment of verapamil in human subjects (Varma et al., 2003). Lastly, combination therapy of dexverapamil and paclitaxel in metastatic breast cancer patients showed increased mean peak paclitaxel concentrations and delayed clearance (Tolcher et al., 1996).

2.3.2 Cyclosporin A

Cyclosporin A is a lipophilic cyclic endecapeptide. The US Food and Drug Administration (FDA) approved cyclosporin A for treatment and/or prevention of transplant rejection, as seen with graft rejection in kidney, liver, heart, lung, and combined heart-lung transplantation. In addition, it is applied in bone marrow transplantation to prevent graft-versus-host disease, as well as in treatment of autoimmune conditions like psoriasis, atopic dermatitis, rheumatoid arthritis, and a variety of glomerular disorders. Cyclosporin A acts by binding to the cytosolic protein, cyclophilin A, the most abundant cyclophilin in T-lymphocytes (Kapturczak et al., 2004). Nephrotoxic effects, both acute and chronic, are among the most common side effects

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of cyclosporin A therapy (Klintmalm et al., 1981). Furthermore, cyclosporin A is a known inhibitor of P-gp (Britten et al., 2000).

Cyclosporin A has a chemical formula of C62H111N11O12 and molecular weight of

1203 g/mol (Figure 2.5). It is a cyclic, highly hydrophobic endecapeptide, and its purified form appears as white prismatic needles, which are neutral and only slightly soluble in water and saturated hydrocarbons. The drug is also slightly soluble in lipids and other organic solvents (Kapturczak et al., 2004). Furthermore, the drug is available in an oral formulation, namely, Sandimmune ®.

Figure 2.5: The chemical structure of cyclosporin A (From: Kapturczak et al., 2004) 2.3.2.2 Pharmacokinetics

Due to its lipophilic properties, the majority of cyclosporin A leaves the blood-stream. The apparent volume of distribution of cyclosporin A varies between 4 and 8 L/Kg (Misteli et al., 1990), while the noncellular fraction of blood cyclosporin A is carried mainly by lipoproteins (Urien et al., 1990). Cyclosporin A is primarily metabolized by CYP3A4 (Shimada et al., 1994) in the liver. CYP3A4 transforms cyclosporin A to more than 30 metabolites by hydroxylation, demethylation, sulfation, and cyclization (Christians and Sewing, 1993), and all metabolites display only minimal, if any, immunosuppressive activity (Radeke et al., 1992). The average half-life of cyclosporin A is approximately 19 hours (Yee, 1991). It is primarily excreted in the bile with less than 1 % contribution of the parent drug, while urinary excretion accounts for 6 % of the oral cyclosporin A dose, of which 0.1 % is unaltered (Maurer and Lemaire, 1986). Furthermore, cyclosporin A crosses the plancenta and is excreted in human milk (Flechner et al., 1985).

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Cyclosporin A influences the pharmacokinetics of sirolimus by increasing its bioavailability via competitive interaction with both CYP3A4 and P-gp (Christians et al., 2003). As corticosteroids are part of most immunosuppressive regimens, they have shown to be substrates, inhibitors and inducers of CYP3A4, as well as potent inducers of P-gp (Salphati and Benet, 1998). Subsequently, they either lower or increase cyclosporin A requirements. Furthermore, cyclosporin A increases the plasma concentrations of atorvastatin and several other statins, probably by OATP1B1 inhibition (Neuvonen et al., 2006).

2.4 Substrates and inhibitors of multidrug

resistance-associated protein 2

2.4.1 Methotrexate

Methotrexate is a folic acid antagonist which is used in a wide array of clinical conditions. It was introduced for the treatment of acute lymphoblastic leukemia in 1948 (Farber and Diamond, 1948), and later on used for cancer monotherapy, and as an antineoplastic and immunosuppressive agent (Mohammad et al., 1979).The drug is also effective for the treatment of psoriasis and rheumatoid arthritis (Oufi and Al-Shawi, 2014). Methotrexate acts by inhibiting the proliferation of malignant cells, primarily by preventing the de novo synthesis of purines and pyrimidines (Chan and Cronstein, 2002). The most common side effects observed after treatment with methotrexate are mucosal ulceration and nausea (Richard et al., 2009, p.461-464). Lastly, methotrexate is a known substrate of multidrug resistance-associated protein 2 (MRP2).

Methotrexate (Figure 2.6) is an orange yellow crystalline powder with empirical formula C20H22N8O5 and molecular weight of 454.4 g/mol (Basile et al., 2002). It is water soluble,

and almost insoluble in alcohol, ether, and chloroform (Chan, 1988).The drug is available in intravenous and oral formulations.

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Figure 2.6: The chemical structure of methotrexate (From: Saxena et al., 2009) 2.4.1.2 Pharmacokinetics

After oral administration, approximately 35 % of methotrexate is bound to plasma proteins and a larger amount is eliminated by the kidneys, whereas less than 10 % of the drug is metabolized to 7-hydroxymethotrexate in the liver (Chiang et al., 2005). Methotrexate is transported by efflux transporters: P-gp, MRPs and breast cancer resistance protein (BCRP) (Yokooji et al., 2007). At doses of 40 mg/m2_{or less, the}

bioavailability of methotrexate is about 42 %, and at doses greater than 40 mg/m2_{it is}

reduced to only 18 %.

Some nonsteroidal anti-inflammatory drugs (NSAIDs) such as salicylate, piroxicam, ibuprofen, naproxen, sulindac, tolmetin, and etodolac, inhibit the renal tubular secretion of methotrexate by inhibiting OATP1 and 3, and MRP2 and 4, thus plasma methotrexate concentrations are increased to levels that can be potentially toxic (El-Sheikh et al., 2006). Furthermore, pantoprazole and omeprazole inhibit methotrexate transport by BCRP, thereby resulting in elevated methotrexate concentrations (Breedveld et al., 2004).

2.4.2 Probenecid

Probenecid is a uricosuric drug that increases uric acid excretion in the urine; therefore, it is primarily used for the treatment of gout and hyperuricemia. Unfortunately, probenecid competitively inhibits the renal excretion of some drugs, thereby increasing their plasma concentrations and prolonging their effects. Other than that, probenecid is

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well tolerated, with only 2 % of patients developing mild gastrointestinal irritation, while 2 % to 4 % of patients may experience mild hypersensitivity reactions (Brunton et al.,

2008).

Probenecid (Figure 2.7) is a white or nearly white, fine, crystalline powder with empirical formula C13H19NO4S and molecular weight of 285.4 g/mol. It is a weak acid (pKa 3.7;

Gutman et al., 2012) and is soluble in dilute alkali, alcohol, chloroform, and acetone, but practically insoluble in water and dilute acids (Xu and Madden, 2011). The drug is available in an oral formulation.

Figure 2.7: The chemical structure of probenecid (From: Himani et al., 2014) 2.4.2.2 Pharmacokinetics

Probenecid has an oral bioavailability of greater than 90 %, is 85 to 95 % bound to plasma albumin, and has a small apparent volume of distribution of 0.003-0.014 L/kg in humans. The maximum adult dose of probenecid is 3 g, and a single oral dose of 2 g (approximately 25 mg/kg) yields peak plasma concentrations of 150-200 µg/ml within 4 hours, while concentrations greater than 50 µg/ml are sustained for 8 hours. Following a 2 g dose, the half-life is 4-17 h, however the half-life is dose-dependent, and decreasing as the dose decreases to 500 mg. Probenecid is metabolized in the liver via oxidation and glucuronidation and is primarily excreted in the urine (75-85 %). Also, probenecid is transported by MRP and the uric acid transporter (Gutman et al., 2012).

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Namkoong et al. (2007) reported that co-administration of probenecid and irinotecan reduced irinotecan-induced late-onset toxicity in the gastrointestinal tissue as they inhibit the biliary excretion of irinotecan by MRP2 inhibition. Probenecid increases plasma concentrations of methotrexate by inhibiting drug efflux mediated by MRP, while at the same time inhibiting folate uptake. It also inhibits the tubular secretion of organic anion derivatives, such as penicillin, by inhibiting organic anion transporters (OATs). The drug is a weak inhibitor of CYP2C19 and blocks the renal transport of many compounds, including many classes of antibiotics, antivirals, and NSAIDs, leading to an increase in their mean plasma elimination half-life that can lead to increased plasma concentrations (Gutman et al., 2012).

Although the available literature provides much information regarding (western) drug-transporter interactions, less is known about potential interactions should natural products be consumed.

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CHAPTER 2 LITERATURE REVIEW: PART III

SCREENING OF PHELA FOR POTENTIAL INTERACTION

WITH MEMBRANE TRANSPORTERS

2.5 An overview on Traditional Medicine

The use of traditional medicines and natural health products is increasing among those living with HIV/ AIDS (Fairfield et al., 1998), one of the reasons being that currently there is no cure for HIV/ AIDS, and the programs used to manage the pandemic are not always satisfactory. Secondly, most traditional medicines have been used for years, hence are often assumed to be safe and efficacious, and are recommended to be used with anti-retroviral treatments (Lekhooa et al., 2010). According to the World Health Organization (WHO), TMs are defined as: “health practices, approaches, knowledge and beliefs incorporating plant, animal, and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination, to treat, diagnose and prevent illness or maintain the well-being”. This is a broad definition that ensures that all types of traditional medicines are included. Other terminologies used in traditional medicine include complementary/alternate medicine (CAM), herbal medicines, and herbs (Crouch et al., 2000).

2.5.1 Interactions with Traditional Medicines

The concomitant use of traditional medicines with prescription drugs may result in potential pharmacokinetic interactions mediated by drug-metabolizing enzymes or transporters, termed herb-drug interactions (Tomlinson et al., 2008; Table 2.1). To date, citrus juices, especially grapefruit juice, have been reported to reduce the bioavailability of orally administered fexofenadine, an antihistamine. This interaction is considered to be caused by the inhibition of intestinal OATPs (Banfield et al., 2002). On the other hand, the interaction of herbal dietary products with transporters has also received increasing attention. For example, repetitive administration of St. John’s wort induces the expression of not only CYP450 enzymes, but also P-gp, which decreases the

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bioavailability of its substrates such as indinavir, cyclosporin A, and digoxin (Durr et al., 2000). It was also demonstrated that extracts of green tea, garlic, and milk thistle inhibit the function of P-gp in vitro (Jodoin et al., 2002). A study conducted by Honda et al. (2004) has reported that grape fruit and orange juice interact not only with P-gp, but also with MRP2, both of which are expressed at apical membranes and limit the apical to basal transport of vinblastine and saquinavir in Caco-2 cells. Therefore, MRP2, in addition to P-gp and CYP3A4, may contribute to the drug pharmacokinetic changes brought on by grape fruit and orange juices. Chiang et al. (2005) reported a life-threatening interaction between methotrexate (substrate of MRP2) and Pueraria lobata root decoction in rats. Here, PLRD significantly decreased elimination and resulted in markedly increased exposure of methotrexate.

Table 2.1: Herb-drug interactions mediated by drug-metabolizing enzymes or

transporters (From: Mills et al., 2005*; Yokooji et al., 2010)

Herb Drug Enzyme/ Transporter Herb-drug interaction

Hypoxide Verapamil CYP3A4/ P-gp* drug toxicity

Sutherlandia Verapamil CYP3A4/P-gp* loss of therapeutic effect Rhei Rhizhoma 2.4-dinitro- MRP2 Increased peak

Phenyl-S- concentration & Area glutathione under the curve of DNP-

(DNP-SG) SG

2.6 Phela

Phela is the name given to the herbal preparation of four African traditional medicinal plants, i.e., Clerodendrum glabrum, Polianthes tuberose, Rotheca myricoides and Senna occidentalis. These plants have been used for decades in wasting conditions and for increasing energy in patients with various disease symptoms including: severe chest problems with pain and coughing; high fevers associated with shivers and headaches; severe loss of weight and appetite; vomiting and diarrhoea; bed-ridden patients with stiff posture, and lip wounds. Phela is currently under development by the

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Medical Research Council (MRC) as an immune booster for patients with a compromised immune system (Lekhooa et al., 2012, p.27-39). Although the mechanism of action of Phela is unknown, an in vivo experiment conducted in rats showed that Phela stimulates or restores cyclosporine induced immune suppression, indicating possible IL-2 activation (Lekhooa et al., 2010). Results of clinical safety studies conducted on 40 healthy male participants revealed that participants reported no major side effects, and it was concluded that Phela is safe and well tolerated (Medical Research Council, Indigenous Knowledge Systems lead programme report, 2009).

2.6.1 Physical properties

Phela extract powder (Figure 2.8) is a brown to light brown coloured powder with uniform particle size (size 90 sieved). It is soluble in water and evaporates at around 105 °C (Medical Research Council, Indigenous Knowledge Systems lead programme report, 2009).

Figure 2.8: Phela extract powder

2.6.2 Oral formulation

The finely ground pre-mixed plant powders are encapsulated in a standardized 350 mg unit dose capsule (Medical Research Council, Indigenous Knowledge Systems lead programme report, 2009).

2.6.3 Pharmacokinetic parameters

The consumption of Phela has been calculated to equate to an adult dose of 1081 mg /70 kg/day which is equivalent to 15.4 mg/kg body weight (Medical Research Council, Indigenous Knowledge Systems lead programme report, 2009). The pharmacokinetic

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parameters of Phela in rats were determined using this 15.4 mg/kg dose of Phela. From the results, the metabolite’s half-life was 3.47±0.35 hours and reached maximum concentration at 4.67±1.15 hours. The concentration at steady state was estimated to be 47.52±5.94 PK-area/L, with no drug accumulation when a once daily dose of Phela is taken (Lekhooa et al., 2012, p.73-80).

2.6.4 Phela-drug interactions

From a previous departmental study it was observed that Phela has no significant effect on the activity of CYP450 isoforms (Medical Research Council, Indigenous Knowledge Systems lead programme report, 2009).

2.6.5 Conclusion

Although Phela has shown not to induce drug interactions via the CYP450 enzyme system, other factors must be taken into consideration concerning the transport of Phela, such as its effect on efflux membrane drug-transporters, and more specifically P-gp and MRP2, which are involved in drug absorption in the gastrointestinal tract. Since Phela is reported to be a potential immune modulator (Lekhooa et al., 2012, p.73-80), it may benefit individuals with compromised immune systems such as HIV/AIDS patients. Therefore, there is a need to understand potential herb-drug interactions of Phela in order to predict its safety and toxicological effects that may occur.

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CHAPTER 3 REVIEW OF ANALYTICAL METHODS

3.1 Review of analytical methods for the determination of

paclitaxel and methotrexate in plasma

3.1.1 Paclitaxel

A number of analytical methods for the quantification of paclitaxel in plasma are described, and among those, high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS-MS) are commonly used. Though LC-MS-MS have many advantages over HPLC, the latter is more convenient for paclitaxel analysis (Yonemoto et al., 2007).

LC-MS-MS methods described by Lian et al. (2013) and Rajender and Narayana (2010) are rapid, sensitive, and highly accurate for determination of paclitaxel in plasma.

However, the method reported by Lian et al. (2013) was advantageous in that it required a small sample volume. Though these methods seemed more appealing than HPLC, they require highly specialized and expensive equipment, which are not available in our set-up, hence had to be dismissed.

Regarding HPLC methods, Martin et al. (1998) described a convenient assay for determination of paclitaxel in plasma. The method utilized docetaxel as internal standard and involved a liquid-liquid extraction with diethyl ether. Unfortunately, the mobile phase of ammonium acetate buffer-tetrahydrofuran resulted in inadequate elution of paclitaxel.

The method reported by Coudoré et al. (1999) was simple, and entailed a rapid single-step liquid-liquid extraction with dichloromethane. The mobile phase of distilled water-methanol resulted in poor paclitaxel elution, and plasma interfered with paclitaxel separation.

Another HPLC assay for paclitaxel was explained by Andersen et al. (2006), which involved a large sample volume of 4 000 µl and also used docetaxel as internal

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standard. Furthermore, it made use of solid phase extraction, and a mobile phase of acetonitrile-sodium phosphate buffer, which showed promising separation of paclitaxel. The reviewed methods could not be solely adopted; however, each method held appealing conditions which were selected as a starting point for method development. This includes: the internal standard from Martin et al. (1998) and Andersen et al. (2006); the analytic column and liquid-liquid extraction from Martin et al. (1998); as well as the mobile phase from Andersen et al. (2006).

3.1.2 Methotrexate

Several methods for methotrexate analysis have been reported. These methods are either expensive or time-consuming such as HPLC, radioimmunoassay (RIA), dihydrofolate reductase inhibition assay, enzyme immunoassay (EIA), fluorescence polarization immunoassay (FPIA), and enzyme multiplied immunoassay (EMI; Lobo and Balthasar, 1999).

An HPLC method for determination of methotrexate in plasma described by Lobo and Balthasar (1999) used a small sample volume of 100 µl, but showed low sensitivity. Uchiyama and co-workers (2012) reported methods which involved the use of post-column photochemical reaction, complex chemicals, and tedious extraction processes. Therefore, the use of HPLC was discarded.

Although RIA methods are sensitive, and proven to be technically simple, they are costly, and require time-consuming experimental procedures. Furthermore, they require antibodies and make use of radioactive material with short shelf-life and inconvenient disposal properties (Howell et al., 1980; Tracey et al., 1983). Enzyme assays such as EIA and FPIA make use of expensive enzymes and antibodies (Al-Bassam et al., 1979; Belur et al., 2001; Jolley et al., 1981), therefore cannot be considered for use.

In the Toxicology Laboratory of the Department of Pharmacology, University of the Free State, EMI has for years been the method of choice for patient therapeutic drug monitoring of methotrexate. It has proved to be accurate and reliable, therefore,

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For the purpose of this study, it was felt appropriate to analyse methotrexate by this method.

3.2 Review of methods for determination of the

protein-binding capacity of paclitaxel

Concerning the determination of free concentration and bound fractions of paclitaxel, various methods are described. These techniques are time-consuming, result in loss of analyte to membranes, produce errors due to protein leakage, and can create a shift in concentrations. Available methods include: ultrafiltration, ultracentrifugation, and equilibrium dialysis (Musteata and Pawliszyn, 2006).

Paál and co-workers (2001) reported a simple ultrafiltration method which was fast and utilized a small sample volume of 990 µl. Unfortunately, it was unreliable in that the binding was not temperature controlled, and the volume of ultrafiltrate was not sufficient for the drug assay.

Ultracentrifugation requires costly equipment, and sedimentation, back diffusion viscosity and binding to plasma lipoproteins in the supernatant fluid during the process, can cause errors in the estimation of the free drug concentration (Barré et al., 1985). The ultracentrifugation method reviewed which was described by Gapud et al. (2004), was complicated and inconvenient, and thus discarded.

A reliable equilibrium dialysis method was described by Brouwer et al. (2000). The method involved the use of a small sample volume of 300 µl, which was dialysed against phosphate buffered saline at 37 °C for 24 hours, in a humidified atmosphere of 5 % carbon dioxide. Thereafter, paclitaxel was quantified by liquid scientillation.

A Slide-A-Lyzer® dialysis method was described by Zhao and co-workers (2010), which utilised a large sample volume of 2 000 µl, and dialysis against phosphate buffered saline at 37 °C and 100 rpm, after which paclitaxel was analysed by HPLC.

In reviewing the methods discussed above, it was concluded that none could be adopted, owing to equipment used. However, Slide-A-Lyzer® equilibrium dialysis, as described by Zhao et al. (2010), was considered, and used as such.

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CHAPTER 4 OBSERVATIONS FROM THE REVIEW

4.1 Observations from the review

 There is a need to develop a model for screening traditional medicines for potential interaction with absorption transporters

 The interaction of herbal dietary products with transporters has recently received increasing attention.

 Phela, a traditional medicine, is under development by the Medical Research Council of South Africa as an immune booster.

 It is important to determine possible interactions between Phela and transporters.  The discovery of membrane drug-transporters has led to renewed interest in,

among others, the mechanism of drug absorption.

 Saturation or inhibition of influx (pump in) transporters leads to decreased drug absorption, while inhibition of the efflux (pump out) transporters leads to increased drug absorption and concentration.

 It is now established that drug-transporters are an important factor in the bioavailability of some drugs, hence are source of drug interactions.

 Knowledge of the possible interactions will help to determine the mechanism of action of Phela on the transporters, making it easier to predict the effects on the transport of Phela out of cells as well as its bioavailability.

4.2 Aim

 To investigate the effect of Phela, a traditional medicine, on drug-transporters P-gp and MRP2, in the gastrointestinal tract of a rat model.

4.3 Objectives

 To develop an HPLC method for the analysis of paclitaxel in plasma.

 Determination of a drug interaction between Phela and paclitaxel in vitro, by a direct drug interaction testing experiment and Slide-A-Lyzer® technique.

 Determining the effect of Phela on the pharmacokinetics of paclitaxel and methotrexate, after oral administration in rats using HPLC-UV and enzyme multiplied immunoassay, respectively.

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CHAPTER 5 DETERMINATION OF PACLITAXEL IN PLASMA BY HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY

Summary

A high performance liquid chromatography (HPLC) method for the determination of paclitaxel in plasma was developed. It involved liquid-liquid extraction of 100 µl plasma, spiked with paclitaxel, extracted with diethyl ether: dichloromethane (2:1), followed by centrifugation. The supernatant was evaporated to dryness under a stream of nitrogen, reconstituted, and 100 µl was injected into the HPLC. The sample was eluted with a mobile phase of sodium phosphate buffer (pH 2): acetonitrile (60:40, v/v) over a C8 (1)

(4.6 X 250 mm) 5 µ analytic column at 1 ml/min and detected by UV at 230 nm. Docetaxel was used as the internal standard. Under these conditions docetaxel and paclitaxel eluted at retention times of 6.595 and 6.038 minutes, respectively. The

average calibration curve (0 – 15 µg/ml) was linear with a regression equation of y = 0.1931x + 0.0705, and correlation coefficient (r) of 0.9973. The method was used

successfully in animal experiments to measure paclitaxel in the plasma of treated rats.

5.1 Introduction

In this chapter, a high performance liquid chromatography assay is described.

5.2 Methods

A. Materials

5.2.1Apparatus

For weighing gram and milligram quantities of reagents and drug standards, a precision balance (SPB 52, Scaltec Instruments, Goettingen, Germany) and analytic balance (AS 220/C/2, Randwag, Random, Poland) were used. A vortex mixer (Vortex Genie 2, Scientific Industries Inc., Bohemia, NY, U.S.A) and micro centrifuge (Minispin, Eppendorf, Hamburg, Germany) were used for mixing and spinning of the samples. Spectrophotometer (Biochrom Libra S12) was used to determine the wavelength of paclitaxel and docetaxel.

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5.2.2 Reagents and chemicals

All standards and chemicals used were of analytic grade. Paclitaxel and docetaxel were obtained from Sigma-Aldrich Inc. (St. Louis, MO. U.S.A). Orthophosphoric acid (H3PO4),

di-sodium hydrogen orthophosphate dehydrate (Na2HPO4.2H2O), and sodium

dihydrogen phosphate monohydrate (NaH2PO4.H2O) were purchased from Merck

laboratories (Darmstadt, Germany).HPLC grade acetonitrile (C2H3N), diethyl ether

((C2H5)2O), dichloromethane (CH2Cl2), ethanol (C2H6O) and methanol (CH4O) were

from Honeywell Burdick and Jackson (Muskegon, MI, U.S.A).Fresh plasma was obtained from healthy volunteers after informed consent.

5.2.3 Chromatographic system

The HPLC system was an Agilent, Hewlett Packard 1100 Series, equipped with an Infinity quaternary pump (Waldbronn, Germany), with a 1260 Infinity degasser attached to a G1313A autosampler (Waldbronn, Germany), and a G1314A UV wavelength detector (Tokyo, Japan). Data was collected using ChemStation software.

5.3 Preliminary experiments

5.3.1 Selection of a mobile phase

Initially, a mobile phase of distilled water (solvent A) and pure acetonitrile (solvent B) was tried in a ratio of 40:60 (A:B), but with little success, as paclitaxel eluted poorly. Similarly, a mobile phase of distilled water (solvent A) and pure methanol (solvent B) in a ratio of 30:70 (A:B) did not yield good results as paclitaxel still eluted poorly.

Thereafter, a mobile phase of 20 mM sodium phosphate buffer at pH 2 (solvent A) and pure acetonitrile (solvent B) was tried with different gradients. Finally, paclitaxel and docetaxel showed satisfactory separation at a ratio of 40:60 (A:B), and the respective peaks were sharp and well resolved. As such, this mobile phase was selected for further evaluation in the subsequent experiments.

The effect of the traditional medicine phela on p-glycoprotein and multidrug resistance-associated protein 2 drug transporters in the gastrointestinal tract of a rat model